REVIEW ARTICLE

The ecology of environmental DNA and implicationsfor conservation genetics

Matthew A. Barnes1 • Cameron R. Turner2,3

Received: 6 April 2015 / Accepted: 31 August 2015

� The Author(s) 2015. This article is published with open access at Springerlink.com

Abstract Environmental DNA (eDNA) refers to the

genetic material that can be extracted from bulk environ-

mental samples such as soil, water, and even air. The

rapidly expanding study of eDNA has generated unprece-

dented ability to detect species and conduct genetic anal-

yses for conservation, management, and research,

particularly in scenarios where collection of whole organ-

isms is impractical or impossible. While the number of

studies demonstrating successful eDNA detection has

increased rapidly in recent years, less research has explored

the ‘‘ecology’’ of eDNA—myriad interactions between

extraorganismal genetic material and its environment—and

its influence on eDNA detection, quantification, analysis,

and application to conservation and research. Here, we

outline a framework for understanding the ecology of

eDNA, including the origin, state, transport, and fate of

extraorganismal genetic material. Using this framework,

we review and synthesize the findings of eDNA studies

from diverse environments, taxa, and fields of study to

highlight important concepts and knowledge gaps in eDNA

study and application. Additionally, we identify frontiers of

conservation-focused eDNA application where we see the

most potential for growth, including the use of eDNA for

estimating population size, population genetic and genomic

analyses via eDNA, inclusion of other indicator biomole-

cules such as environmental RNA or proteins, automated

sample collection and analysis, and consideration of an

expanded array of creative environmental samples. We

discuss how a more complete understanding of the ecology

of eDNA is integral to advancing these frontiers and

maximizing the potential of future eDNA applications in

conservation and research.

Keywords Environmental DNA � Metabarcoding �Metagenetics � Metagenomics � Monitoring � Surveillance

Introduction

Conservation and ecological understanding have benefitted

from recent, rapid advancements in non-invasive genetics,

the analysis of genetic material within traces of organisms

such as hair, feces, and other shed biological materials,

rather than whole organisms (Beja-Pereira et al. 2009). One

developing advancement in non-invasive genetic methods

is the study of environmental DNA (eDNA), which refers

specifically to the analysis of genetic material collected not

through targeted methods such as deploying fur traps or

collecting fresh scats, but extracted from bulk environ-

mental samples such as soil, water, or air (Taberlet et al.

2012). Rapid advances in technology and concurrent

declines in cost have landed eDNA on a recent annual

survey of global conservation horizons (Sutherland et al.

2013) while others herald a future of ‘‘conservation in a

cup of water’’ (Lodge et al. 2012). While the first analyses

of genetic material from environmental samples—and

indeed, the first use of the term environmental DNA—

occurred within the field of microbiology, where

researchers extracted DNA directly from marine sediments

& Matthew A. Barnes

matthew.a.barnes@ttu.edu

Cameron R. Turner

crt343@ecosysgen.com

1 Department of Natural Resources Management, Texas Tech

University, Lubbock, TX 79409, USA

2 Department of Biological Sciences, University of Notre

Dame, Notre Dame, IN 46556, USA

3 ecoSystem Genetics LLC, South Bend, IN 46635, USA

123

Conserv Genet

DOI 10.1007/s10592-015-0775-4

to characterize the microbial communities contained within

(Ogram et al. 1987), more recently, similar methods have

been applied as a tool for the study and conservation of

macrobial communities. Over time, many creative methods

have been developed for the ‘‘sight-unseen’’ detection of

organisms (sensu Jerde et al. 2011). Today, diverse fields

of biological and environmental study use DNA to detect

various taxa across many different types of environments,

including forensics (van Oorschot et al. 2010), fecal pol-

lution tracking (Caldwell et al. 2011), paleogenetics (Ped-

ersen et al. 2015), and environmental biosafety (Nielsen

et al. 2007). Detection and analysis of eDNA have been the

topic of several recent literature reviews (Blanchet 2012;

Dıaz-Ferguson and Moyer 2014; Rees et al. 2014b; Boh-

mann et al. 2014), including one focused on the conser-

vation biology implications of eDNA (Thomsen and

Willerslev 2015). However, the present review uniquely

examines the conservation applications of eDNA through a

lens we call ‘‘the ecology of eDNA.’’

The ecology of eDNA includes its origin, state, trans-

port, and fate within the environment. Understanding the

origin of eDNA- the source of an organism’s genetic

material shed into its environment and the factors influ-

encing its production- can inform our understanding about

the taxa and environments for which eDNA represents an

effective conservation and research tool. Understanding the

state of eDNA—characterizing the mutable forms of eDNA

after it is shed from an organism and as it moves through

the environment—provides insight into its capture and

analysis. eDNA is transported through the environment

after it is shed from an organism, and understanding this

transport is essential for relating detected eDNA to species

presence in space and time. Finally, understanding the fate

of eDNA—how it degrades and what factors influence

degradation—also improves spatiotemporal inferences of

species presence from eDNA. To illustrate this framework,

we review key findings of eDNA studies from diverse

environments, taxa, and fields. An exhaustive review of the

ever-increasing list of relevant literature pertaining to DNA

detection of species is nearly impossible; however, we

highlight cases across many fields that can inform con-

servation and study of extant macrobiota. We intentionally

exclude intraorganismal eDNA (e.g., microbial eDNA)

from our scope because its ecology is not different from

that of the organisms themselves. In contrast, we argue that

extraorganismal eDNA interacts with its environment in

unique ways that matter for conservation applications.

Following our development of the ecology of eDNA

framework, our review concludes with frontiers of con-

servation-focused eDNA applications in which we see the

most potential for growth, including the use of eDNA for

estimating population size, population genetic and genomic

analyses via eDNA, inclusion of other indicator

biomolecules such as environmental RNA or proteins,

automated sample collection and analysis, and considera-

tion of a wide array of creative environmental samples. A

more complete understanding of the ecology of eDNA will

contribute to the advancement of these frontiers and the

maximization of additional future eDNA applications.

Before exploring the ecology of eDNA in detail, we briefly

review its previous conservation applications.

A brief history of environmental DNAin conservation

As a conservation tool, application of eDNA technology

has focused primarily on the detection of target species,

especially in cases where traditional sampling strategies

may fall short. For example, early detection and rapid

response represent cornerstones of effective management

for invasive species (Lodge et al. 2006); however, despite

the propensity of invasive populations to reach large pop-

ulation sizes with disruptive and obvious negative impacts,

the initial propagules in an incipient invasion, as well as

individuals at the leading edge of an invasion front, are rare

and therefore difficult to detect. Thus, one of the first

demonstrations of macrobial eDNA surveillance targeted

invasive American bullfrogs (Rana catesbeiana) in French

wetlands (Ficetola et al. 2008). The high-profile invasion of

bigheaded carps Hypophthalmichthys molitrix and H.

nobilis into waters of the midwestern United States (USA)

has also represented a frequent target of eDNA surveillance

efforts (Jerde et al. 2011; Mahon et al. 2013; Turner et al.

2014b). Other eDNA surveillance targets in natural waters

have included Bluegill Sunfish (Lepomis macrochirus)

invading ponds in Japan (Takahara and Minamoto 2013),

New Zealand mudsnails (Potamopyrgus antipodarum) in

streams of Idaho, USA (Goldberg et al. 2013), Ponto-

Caspian zebra mussels (Dreissena polymorpha) in the

midwestern USA (Egan et al. 2013), Louisiana crayfish

(Procambarus clarkii) in France (Treguier et al. 2014), and

African Jewelfish (Hemichromis letourneuxi) and Burmese

pythons (Python bivittatus) in Florida, USA’s ponds

(Moyer et al. 2014) and wetlands (Piaggio et al. 2014),

respectively.

In addition to detecting invasive species in natural sys-

tems, conservation efforts have also been bolstered by the

application of genetic surveillance methods to detect

potential invaders in transit. For example, multiple studies

have proposed the use of eDNA to detect invasive organ-

isms within ballast waters of transoceanic ships (Li et al.

2011; Mahon et al. 2012; Egan et al. 2013). Similar assays

have successfully identified benthic invertebrates and their

resting stages within ballast tank sediments (Darling and

Tepolt 2008; Harvey et al. 2009; Briski et al. 2011). eDNA

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monitoring has also been proposed to assess authenticity of

imported ornamental fish and identify potential invasive

contaminant species within shipments (Collins et al. 2012)

as well as within the trade of bait associated with recre-

ational fishing (Mahon et al. 2014).

Threatened and endangered species have represented

another common target for eDNA application because their

rarity makes them difficult to observe and legal restrictions

can limit even routine handling of such species through

more traditional methods such as trapping (Thomsen et al.

2012a). For example, eDNA assays have been used to

detect the eastern hellbender (Cryptobranchus a. alle-

ganiensis), an amphibian of high conservation concern, in

Indiana and Missouri, USA (Olson et al. 2012). In the UK,

eDNA assays have been validated for detection of the

threatened great crested newt Triturus cristatus (Rees et al.

2014a). Sigsgaard et al. (2015) reported that eDNA surveys

for endangered fish in Denmark outperformed traditional

methods. In the western USA, eDNA techniques have been

optimized for detection of endangered Bull Trout Salveli-

nus confluentus (Wilcox et al. 2013, 2014), and other

efforts have targeted rare marine mammals in the Baltic

Sea (Foote et al. 2012).

Bioassessment, the characterization of ecosystem health

through the measurement of local ‘‘indicator’’ organisms, is

another conservation application which has benefitted from

the integration of eDNA technology. For example, eDNA

collected in terrestrial soil samples have been used to

assess earthworm diversity which can relate to healthy

ecosystem function (Bienert et al. 2012). Yoccoz et al.

2012 suggested that soil eDNA could also enable

unprecedented rapid assessment of local plant communities

and community-level responses to climate change. In terms

of detection capabilities, the success of eDNA monitoring

in aquatic environments has compared well with more

traditional kick net sampling of benthic macroinvertebrates

in Switzerland streams (Machler et al. 2014). Furthermore,

advancing technologies at decreasing costs hold the

potential to make eDNA detection of a variety of indicator

species (i.e. benthic invertebrates, fish, algae) faster and

more cost effective than traditional surveys (Stein et al.

2014). We will provide additional examples as we proceed

with the current review.

Understanding the ecology of environmental DNAto improve conservation

The rapid advancement of eDNA-based conservation

applications and potential contributions to research are

exciting; however, capturing and identifying eDNA as well

as interpreting the results of these efforts will benefit from

a more complete understanding of the ecology of eDNA

(i.e. the origin, state, transport, and fate of eDNA mole-

cules; Fig. 1). Research from many fields, including mac-

robial eDNA detection, microbiology, and water quality

monitoring using fecal indicator bacteria, can all contribute

to advancements in our understanding (Barnes et al. 2014).

Below, we outline a framework consisting of four major

questions to help guide research aimed at elucidating the

ecology of eDNA.

Origin: What are the physiological sources of eDNA

production?

Despite burgeoning interest and study of eDNA applica-

tions outlined above, the physiological origins of the

material collected as eDNA remain uncertain (Fig. 1a).

Microbial research that pioneered detection of genetic

materials in environmental samples recognized that eDNA

was present in both intracellular and extracellular forms

(Ogram et al. 1987). It seems likely that multicellular

organisms shed genetic material into their environment first

as sloughed tissues and whole cells, and then those cells

break down and release DNA into the environment.

However, uncertainty remains in regards to the origins of

sloughed materials. One of the earliest studies to target

detection of vertebrate eDNA collected terrestrial mammal

mitochondrial eDNA from aquatic environments, impli-

cating fecal origins of such material (Martellini et al.

2005). Although feces remains a probable source of eDNA

from a wide range of taxa targeted in aquatic (Thomsen

et al. 2012a) and terrestrial environments (Andersen et al.

2012), high rates of success detecting taxa which produce

slimy coatings such as amphibians (Ficetola et al. 2008)

and fish (Jerde et al. 2011) suggest that other bodily fluids

also act as a source of eDNA. Still other studies have

demonstrated that dead carcasses and predator feces may

also serve as an eDNA source in some cases (Merkes et al.

2014). Although some research has examined the size

distribution of eDNA-bearing particles in aquatic envi-

ronments to provide clues to its possible origins (Turner

et al. 2014a), we are unaware of any microscopy-based

examinations or other studies that directly address the

question of what comprises eDNA collected for research

and conservation.

Although the physical identity of the material compris-

ing eDNA remains relatively unstudied, research efforts

have demonstrated that many factors can influence the

amount of genetic material released by organisms into their

environments. For example, in an aquarium study of Idaho

giant salamanders (Dicamptodon aterrimus), rate of eDNA

production related positively to salamander biomass (Pil-

liod et al. 2014). A similar trend of eDNA production

positively related to biomass was observed in Bluegill

Sunfish (Maruyama et al. 2014). However, when corrected

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for biomass, juvenile Bluegill Sunfish released eDNA at a

higher rate than adults (target amplicon copies per hour per

gram fish body weight), leading the authors to conclude

that ontogenetic factors such as changes in behavior and

metabolism influence eDNA production. Indeed, Klymus

et al. (2014) found that increased feeding behavior of

bigheaded carps increased eDNA shedding rates. Finally,

beginning a trend that will emerge throughout our discus-

sion of the major research questions facing future eDNA

studies, local abiotic and biotic environmental conditions

can influence eDNA production. For example, temperature

and microbial activity contributed to DNA release from

plant matter in freshwater sediments (Pote et al. 2009a).

Understanding the origin of eDNA—the source of the

genetic material an organism sheds into its environment

and what factors influence eDNA production- will inform

our understanding about the taxa and environments for

which eDNA will represent an effective conservation and

research tool (Fig. 2a). For example, increased knowledge

about the relationship between eDNA production and

organism size, age, and/or biological activity can guide

eDNA collection. Conservation efforts utilizing eDNA

may be able to maximize success by taking into account

temporal (e.g. seasonal events such as mating or die-offs)

and spatial (e.g. diel or other cyclic migrations) patterns to

target sampling windows in which genetic materials

accumulate. On the other hand, relative differences

between species or age classes within a species will help

identify populations for which eDNA detection represents a

sensitive and accurate detection tool and therefore useful

for research and conservation.

State: In what physical forms does eDNA exist?

Related to the origin of eDNA is the question of its state

within the environment (Fig. 1b). We previously noted that

studies have found eDNA in the environment in both

intracellular and extracellular forms. Presumably, eDNA

transitions over time from intracellular to extracellular, and

indeed considerable knowledge exists on the various eco-

logical processes driving this transition (Levy-Booth et al.

2007). However, few studies have considered the state of

what is collected during an eDNA survey. A notable

exception is the work of Turner et al. (2014a) who looked

at the particle size distribution of Common Carp eDNA in a

small lake and zoo pond using serial filtration of water

samples through filters of decreasing pore sizes

(180–0.2 lm) and concluding with an ethanol precipita-

tion. They observed Common Carp eDNA across all size

fractions, suggesting that Common Carp eDNA was col-

lected as aggregations of cells on filters with larger pore

sizes and extracellular DNA in the final precipitation (i.e.

particle sizes \0.2 lm). The largest percentage of total

Common Carp eDNA recovered occurred within the

1–10 lm size fraction, but further size fractionation studies

are needed to determine whether the findings of Turner

et al. (2014a) represented a general trend across all eDNA

studies or a taxa- or environment-specific phenomenon.

egestionexcretion

reproductiondecomposition

predators

exfoliation

intramembranous

extramembranous

particulate

free / dissolved

enzymatic degradation

mechanical fragmentation

chemical degradation

radiation degradation

diffusion

advectionresuspensionbiological transportanthropogenic transport

scavengerscoprovoresdetritivores

Primary Secondary

horizontal gene transfer

secretion

settling

collect

capture

extract

assayinhibition

contamination

low quantity

analyze

designpatchiness

PCR artifacts

Seq. artifacts

fragmentation

Challenges

Technical

species ID

STATEORIGIN

FATE TRANSPORT

A B

CD

THE ECOLOGY of eDNA

E

Fig. 1 Processes and properties within four domains of eDNA ecology (a–d) and key technical challenges (e) can guide eDNA conservation and

research applications

Conserv Genet

123

With an apparent lack of systematic comparison between

eDNA capture methods to date (but see Deiner et al. 2015),

trial and error or logistical constraints have dominated the

field thus far (Turner et al. 2014a). Understanding the state

of eDNA (i.e. characterizing the mutable forms of eDNA

after it is shed from an organism and as it moves through the

environment) will provide critical insight into its capture

and analysis (Fig. 2b). For example, specific filter sizes or

capture methods may be targeted to specific eDNA particles

of interest based upon knowledge of the system under

consideration. Furthermore, an improved understanding of

the size distribution of not only target eDNA particles but

also non-target eDNA and other particles responsible for

PCR interference or inhibition could warrant use of multi-

stage filtration or eDNA capture processes (e.g. ‘‘pre-fil-

tration’’ of samples sensu Turner et al. 2014a). Knowledge

of the size distribution of various particles in the aquatic

environment will also inform navigation of the trade-off

which exists between filter pore size and amount of water

that can be processed before filter clog; smaller filter pore

sizes capture more particles from the filtrate but limit fil-

tration volume and speed while larger filter pore sizes filter

larger volumes of water more quickly but may allow

smaller target particles to pass through. A more thorough

knowledge of the size distribution of target and non-target

particles will permit selection of filter sizes and sampling

strategies that optimally navigate sampling sensitivity and

efficiency. The size of eDNA-bearing particles will also

influence horizontal and vertical transport in the environ-

ment, aggregation and disaggregation dynamics, and con-

sumption by other organisms (Turner et al. 2014a).

Transport: How does eDNA physically move

through the environment?

After being shed from an organism, eDNA moves through

its environment, which could influence the inferences of

eDNA-based research and conservation (Fig. 1c). Much of

the early work informing eDNA transport was inspired by

concern over the transgenes of genetically modified organ-

isms, especially agricultural crops, transferring to wild

bacteria or other organisms. For example, Douville et al.

(2007) sampled water to demonstrate that a gene from Bt

corn could leach from the terrestrial into the aquatic envi-

ronment and be transported many kilometers downstream.

While the movement of plant material from an agricultural

field into a nearby waterway is expected, a more surprising

study in Switzerland found terrestrial plant eDNA in

groundwater at a depth of 3.2 m and in groundwater-fed

artesian fountains (Pote et al. 2009b). In a more direct study

of the downstream transport potential of DNA, Foppen et al.

(2011) generated synthetic DNA tracers for an injection

experiment in two streams in the Netherlands. They

observed that the physical movement of artificial DNA

tracers matched the speed of standard NaCl tracers, but

considerable reductions in DNA quantity with downstream

fast decay

slow decay

time

[eD

NA

]

time

[eD

NA

]

true

[eD

NA

][e

DN

A]

reproduction

decomposition

watersample

falsewatersample

filter type A

filter type B

[eD

NA

][e

DN

A]

parti

cle

size

rang

e

STATEORIGIN

FATE TRANSPORT

A B

CD

THE ECOLOGY of eDNA

Fig. 2 eDNA ecology affects population inferences. a eDNA from

reproduction and decomposition could produce similar temporal patterns

despite different origins. b Different filter types could yield different

eDNA concentrations that reflect particle size classes rather than

population size differences. c Resuspension of old sedimentary eDNA

could produce false inferences of presence after organisms are gone.

dDifferent environmentally-mediated eDNA decay rates could confound

inferences about population size or biomass from eDNA concentration

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movement suggested that adsorption, biological uptake, and

other interactions between the DNA molecules and the

stream environments were common. Even so, the artificial

DNA tracer remained detectable at the furthest sampling site

1192 m downstream (Foppen et al. 2011).

As we’ve discussed, extracellular eDNA like the artificial

tracer of Foppen et al. (2011) is just one component of what is

detected in eDNA-based conservation and research applica-

tions. It is likely that eDNA represents a complex mixture of

particles ranging from extracellular DNA molecules up to

whole cells and aggregations of cells (Turner et al. 2014a).

More recently, studies have begun to examine how this com-

plex DNA signal is transported through aquatic environments.

For example, using eDNA from two invertebrate species,

researchers observed horizontal (i.e. downstream) eDNA

transport up to 12.3 km (Deiner and Altermatt 2014). Vertical

transport (i.e. settling) of fish eDNA and accumulation in pond

and river sediments was recently described by Turner et al.

(2015). The high concentrations of sedimentary fish eDNA they

observed also suggest that resuspension of settled eDNA rep-

resents an important element of eDNA ecology. Overall,

understanding this transport is essential to relating detected

eDNA to species presence in both space (i.e. how close was a

species to the site of eDNA detection) and time (i.e. how

recently was the detected species present). In the case of

important conservation applications, being able to connect a

positive eDNA detection within specific spatial and temporal

boundaries is essential to drawing robust conclusions (Fig. 2c).

Fate: What factors influence eDNA persistence?

Although the detection of ancient DNA in terrestrial

(Willerslev et al. 2003; Lydolph et al. 2005; Haile et al. 2009;

Hebsgaard et al. 2009; Jørgensen et al. 2012) and aquatic

(Matisoo-Smith et al. 2008; Anderson-Carpenter et al. 2011;

Pedersen et al. 2013; Stager et al. 2015) sediments as well as

frozen ice cores (Willerslev et al. 2007) testifies to the fact

that eDNA from diverse taxa can remain in the environment

for a very long period of time under certain conditions, DNA

possesses limited chemical stability (Lindahl 1993); in most

cases, as soon as it is shed from an organism eDNA begins to

degrade (Fig. 1d). Factors which influence the persistence of

eDNA in aquatic environments were the focus of a recent

review which considered studies across a wide range of

disciplines, including detection of macrobial eDNA,

microbiology, and water quality assessment via detection of

fecal indicator bacteria (Barnes et al. 2014). The persistence

of eDNA in terrestrial environments has also been the topic

of recent reviews (Nielsen et al. 2007; Levy-Booth et al.

2007). These previous reviews have concluded that the

factors which influence eDNA persistence fall into three

broad categories: DNA characteristics (i.e. conformation,

length, and association with cellular/organellar membranes);

the abiotic environment (i.e. light, oxygen, pH, salinity, and

the abundance and composition of substrates); and the biotic

environment (i.e. the composition and activity of the

microbial community and extracellular enzymes).

A summary of major findings related to eDNA persistence

is presented in Table 1. As was the case with eDNA transport,

much of study of eDNA persistence has been motivated by

concern over the release of transgenes from genetically

modified organisms. Such studies have examined persistence

of genes from transgenic crops in terrestrial soils (Widmer

et al. 1996, 1997; Hay et al. 2002; Gebhard and Smalla 2006)

and nearby groundwater and riverine environments (Matsui

et al. 2001; Zhu 2006). Across these studies, transgenic

material remained detectable for long periods of time (days to

years) in terrestrial soils, but much shorter periods of detec-

tion (hours to days) occurred in aquatic environments

(Table 1). More recent studies have focused on eDNA with

animal origins. Much of this work has focused on aquatic

habitats in laboratory (Dejean et al. 2011; Thomsen et al.

2012b; Goldberg et al. 2013; Barnes et al. 2014; Strickler

et al. 2015), while several studies have occurred in aquatic

field settings (Thomsen et al. 2012a; Merkes et al. 2014).

Turner et al. 2015 presented one of the first comparisons of

accumulation of eDNA in open-water versus aquatic sedi-

ment samples. Fewer studies have examined eDNA persis-

tence in terrestrial environments (Andersen et al. 2012). In

terms of quantifying eDNA persistence, pioneering manipu-

lative studies by Barnes et al. (2014) and Strickler et al.

(2015) have measured co-varying environmental conditions

in an attempt to understand the drivers of eDNA degradation

rates. Recent studies by Barnes et al. (2014), Pilliod et al.

(2014), and Strickler et al. (2015) all suggest that environ-

mental conditions play an integral role in the fate of eDNA

once it has been shed from an organism.

Overall, the wide range of persistence times in the lit-

erature suggest that there is still much to learn about eDNA

degradation. An improved understanding of eDNA persis-

tence will benefit our ability to interpret the results of

eDNA detection because, as we argued in our previous

discussion of eDNA transport through the environment, an

understanding of eDNA persistence is critical to delimiting

the proximity of a detected organism in space and time

(Fig. 2d). Uncovering this knowledge will likely require

more research on eDNA itself as well as the interactions

between eDNA and its environment.

Future conservation applications of environmentalDNA

In addition to increased understanding of the ecology of

eDNA, the accuracy and sensitivity of genetic analysis

technology are improving rapidly and commensurately

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Table 1 Summary of major findings on eDNA persistence

Environment eDNA target Major findings Citation

Terrestrial soil Bactrian camel (Camelus bactrianus) eDNA could be detected in soil samples from a

Danish zoo pen where the species had not been

present for 6 years

Andersen

et al.

(2012)

Laboratory freshwater

aquaria

Common carp (Cyprinus carpio) eDNA concentration decreased exponentially;

after 4 days the probability of detection was

\5 %. Rare detections occurred as late as

14 days. Degradation rate negatively correlated

with indices of microbial activity (biochemical

oxygen demand, total DNA concentration, and

chlorophyll a concentration) and pH

Barnes

et al.

(2014)

Laboratory freshwater

mesocosms and small

natural ponds

American bullfrog (Rana catesbeiana) and

Siberian Sturgeon (Acipenser baerii)

eDNA remained detectable with[5 % probability

for 25 days in glass beakers, while fish eDNA in

small ponds demonstrated[5 % detection

probability for 17 days

Dejean

et al.

(2011)

Terrestrial soil Transgenic sugar beet litter (Beta vulgaris) eDNA remained detectable for 2 years Gebhard

and

Smalla

(2006)

Laboratory freshwater

aquaria and eutrophic 5th

order river

New Zealand mudsnail (Potamopyrgus

antipodarum)

eDNA remained detectable for 21 days following

snail removal

Goldberg

et al.

(2013)

Permeable bags in

terrestrial soil

Transgenic poplar leaf material (Populus alba

L. 9 Populus grandidentata Michx.)

eDNA remained detectable for no longer than

4 months

Hay et al.

(2002)

Lentic environment Exogenous plasmid pEGFP (Clontech, USA) Plasmid DNA was undetectable within 170 h in

untreated water, while no degradation was

observed in the presence of antimicrobial agent

EDTA. Culling the bacterial community via

filtration also resulted in reduced DNA

degradation rates

Matsui

et al.

(2001)

Mesocosms floated in

experimental ponds and

laboratory freshwater

aquaria

Silver Carp (Hypophthalmichthys molitrix)

carcasses, fecal samples of eagles (Haliaeetus

leucocephalus) which had eaten Silver Carp

DNA could be recovered from swabbed carcasses

for up to 28 days of areal environmental

exposure, independent of ambient temperature,

humidity, precipitation, and UV exposure. Silver

Carp eDNA was also detected from fecal samples

of birds who had eaten Silver Carp, and

detections occurred from feces which had been

deposited up to 30 days prior

Merkes

et al.

(2014)

Laboratory freshwater

aquaria

Idaho giant salamander (Dicamptodon

aterrimus)

eDNA decreased exponentially in light and shaded

treatments but was no longer detectable in full-

sun treatments after 8 days, detectable in shaded

treatments after 11 days, and detectible in

refrigerated controls after 18 days

Pilliod

et al.

(2014)

Laboratory freshwater

mesocosms

Bullfrog (Lithobates catesbeianus) eDNA decreased exponentially but remained

detectable\1–54 days following organism

removal. Higher temperatures and lower pH

decreased degradation rates. UV-B intensity

interacted with other factors but appeared to have

a positive relationship with eDNA degradation

Strickler

et al.

(2015)

Laboratory marine aquaria European Flounder Platichthys flesus and

Three-spined Stickleback Gasterosteus

aculeatus

eDNA from two marine fish species decayed

exponentially, resulting in failure to detect eDNA

after 0.9 and 6.7 days

Thomsen

et al.

(2012a)

Outdoor freshwater

mesocosms

Common spadefoot toad Pelobates fuscus and

great crested newt Triturus cristatus

eDNA persisted 7–14 days following removal of

live organisms

Thomsen

et al.

(2012b)

Experimental ponds Bighead and Silver Carp (Hypophthalmichthys

spp.)

eDNA was observed in pond sediments for

[130 days after fish removal, but was not

detected in pond water after the same period

Turner

et al.

(2015)

Conserv Genet

123

with increasing affordability (Shokralla et al. 2012).

Although a thorough discussion of the growing technical

and methodological considerations of eDNA applications is

beyond the scope of the present review, ‘‘Appendix’’ pro-

vides an overview of important considerations in study

design, sample collection and preparation, eDNA assays,

and analysis of eDNA data. Most of the examples of eDNA

conservation applications we have presented so far—and

indeed most of the macrobial eDNA studies to date—have

focused on single-species, presence/absence monitoring

with established technologies like endpoint PCR and

quantitative PCR (qPCR). Given continuing advancement,

a recent perspective piece lauded the potential of expand-

ing the use of eDNA for addressing an expanded list of

ecological issues, including diet characterization, descrip-

tion of trophic interactions, and whole-ecosystem biodi-

versity monitoring (Yoccoz 2012). Here, we outline seven

frontiers of eDNA conservation applications representing

the cutting edge or intriguing future applications of eDNA

technology.

Description of whole communities

Although the examples we have identified thus far repre-

sent applications targeting single species, many researchers

have begun to identify multiple species concurrently by

using taxonomically general PCR primers paired with

cloning and Sanger sequencing (Minamoto et al. 2012) or

high-throughput sequencing (HTS; Thomsen et al. 2012b).

These efforts have been referred to as metagenomics,

metagenetics, metasystematics, or metabarcoding (Taberlet

et al. 2012). Ongoing debate about the most apt name for

this effort notwithstanding (Esposito and Kirschberg 2014),

the ability to use eDNA to detect many species simulta-

neously inspires considerable conservation appeal (Yoccoz

2012). The ability to survey multiple species within a

single sampling effort using eDNA makes surveys more

efficient and economical. Furthermore, the use of increas-

ingly general primers or shotgun sequencing (i.e. without

taxonomically general PCR) opens up the possibility that

managers and researchers do not need to choose target

organisms a priori, thus facilitating detection of unexpected

endangered or introduced species. Finally, species inter-

actions represent an important consideration for conserva-

tion efforts, and understanding the distributions of multiple

interacting species in a single survey could contribute to

the maintenance of intact communities.

Many studies applying eDNA to the description of

groups of organisms or whole communities have begun to

emerge. For example, one study used HTS to distinguish

multiple earthworm species in soil samples and speculated

that similar methods could be used to characterize other

soil-dwelling taxa (Bienert et al. 2012). Indeed, other

groups of taxa that have successfully been the target of

eDNA studies in soil samples include nematodes (Po-

razinska et al. 2010; Vervoort et al. 2012) and boreal plant

communities (Yoccoz et al. 2012). Sequencing of genetic

material recovered from leaf litter samples has reflected

terrestrial arthropod diversity in China and Vietnam (Yang

et al. 2014). Other examples of whole-community eDNA

analyses in aquatic environments include survey of fish

species in the Monterey Bay Aquarium, California, USA

(Kelly et al. 2014b), survey of fish species in aquaria and

the Yuma River in Japan (Minamoto et al. 2012), and the

detection of fish and bird eDNA in marine water samples in

Denmark (Thomsen et al. 2012b). Most recently, research

on this frontier has begun to address the substantial

uncertainties and artifacts inherent to using PCR and HTS

on eDNA, which often exists in trace quantities within the

environment (Nguyen et al. 2015). These include false-

positives and false-negatives (Ficetola et al. 2014), PCR

and sequencing errors (Schnell et al. 2015), marker

Table 1 continued

Environment eDNA target Major findings Citation

Laboratory soil mesocosms Transgenic tobacco leaf material and purified

plasmid DNA from Nicotina tabacum cv.

Xanthi

Purified plasmid DNA remained detectable in soil

after 40 days; the same plasmid within ground

transgenic tobacco leaves was detectable after

120 days. Both trials demonstrated rapid initial

degradation followed by long tails of detection at

low levels

Widmer

et al.

(1996)

Terrestrial soil Transgenic tobacco leaf material (Nicotina

tabacum cv. Xanthi)

eDNA signal of leaf material buried in 10 cm soil

declined rapidly over 14 days but remained

detectable up to 77 days

Widmer

et al.

(1997)

Groundwater and riverine

environments

Transgenic Bt (Bacillus thuringiensis) corn

plasmid DNA (Zea mays)

Plasmid DNA degraded to undetectable levels

within 48–96 h in aquatic environments;

however, eDNA remained detectable throughout

a 192 h experiment when water was sterilized by

autoclave

Zhu

(2006)

Conserv Genet

123

selection bias (Deagle et al. 2014), and bioinformatic

processing artifacts (Rossberg et al. 2014). When uncer-

tainty is handled transparently and rigorously, HTS can

obtain robust biodiversity information from eDNA and

provide reliable inferences for conservation.

Estimation of organism abundance

Following many proofs-of-concept of species detection

with eDNA, a logical next question to emerge is what

information besides presence can be acquired using eDNA.

One promising line of inquiry using eDNA is asking

whether abundance or concentration of eDNA in environ-

mental samples relate to organismal abundance. For

threatened and endangered species with regulations on

their accessibility and handling by researchers and man-

agers, quantification with eDNA could provide otherwise

unobtainable data. For these and additional species that are

difficult to observe, eDNA quantification could provide

clues to habitat use and preference, thus identifying spatial

conservation priorities such as home ranges and dispersal

corridors.

Numerous studies have begun to explore quantification

of eDNA as a means of estimating population size or

biomass. For example, a study of Common Carp in a

Japanese lagoon suggested that Common Carp eDNA

concentration related to fish abundance (Takahara et al.

2012). Meanwhile, a Minnesota, USA lake study of

Common Carp found similar results, with rate of detection

and eDNA concentration correlating positively with fish

abundance (Eichmiller et al. 2014). Similar correlations

have also been found with multiple amphibian species in

Idaho streams (Pilliod et al. 2013) and European ponds

(Thomsen et al. 2012a). Metagenetic analysis of soils at

zoos and farms (i.e. locations where local species compo-

sition is well-known) also reflected community composi-

tion and relative biomass of local vertebrate species

(Andersen et al. 2012). Distinguishing between eDNA

signals of organism abundance and organism proximity (in

both space and time) represents an emerging challenge for

research along this frontier. Water and air can rapidly

transport eDNA across long distances (Deiner and Alter-

matt 2014; Kraaijeveld et al. 2015), and eDNA decays

exponentially in the environment (Barnes et al. 2014).

Thus, models that include eDNA production, transport, and

decay may improve the ability to infer organism abundance

from eDNA quantity.

Population genetics and genomics

Beyond presence/absence and abundance information, is

there more information to be gained from eDNA surveys?

The study of population genetics with eDNA samples

represents one exciting possibility. Advances in noninva-

sive genetic methods, such as the collection and population

genetic analysis of hair, feathers, eggshells, feces, and

other samples have been lauded by the wildlife research

and conservation community (Beja-Pereira et al. 2009).

However, the leap in complexity when moving from

sampling specific materials (e.g. trapped clumps of fur) to

the analysis of mixed genetic materials of various quality

contained within bulk environmental samples used in

eDNA analyses portends considerable hurdles to popula-

tion genetic and genomic analyses. The same complexity

differential applies between, for example, pollen from one

plant and a pollen mixture collected by insects.

Whereas previous, primarily metazoan, eDNA assays

have depended almost exclusively upon species identifi-

cation based on mitochondrial DNA (mtDNA), conserva-

tion interest in identifying and distinguishing species

hybrids, populations, evolutionarily significant units, and

individuals based on eDNA will require improved resolu-

tion before eDNA methods can adequately assess fine

levels of genetic variation or estimate population genetic

measures such as effective population size or genetic fix-

ation. Decay and fragmentation of extraorganismal DNA

presents one challenge, but noninvasive genetics and

paleogenetics have demonstrated remarkable persistence of

short nucleic acids (Hofreiter et al. 2001). Perhaps a greater

challenge arises from the high probability that multiple

individuals and species contribute DNA to any given

environmental sample. Such environmentally pooled DNA

from an unknown number of individuals complicates dis-

tinguishing between individual organisms and estimating

classical population genetic parameters, particularly those

that rely on allele frequency (Toulza et al. 2012). Standard

tools for generating population genetic data also are not

generally designed with the high sensitivity and specificity

necessary to isolate single-species markers from a complex

multi-species mixture. Anecdotally, we have had limited

success amplifying \300 bp microsatellite markers for

bigheaded carp (Hypophthalmichthys spp.; Guo et al. 2013)

from experimental pond eDNA extracts in which their

mtDNA measured ca. 800 copies/lL (Cameron Turner,

unpublished data). Inhibition, off-target primer binding,

and low nuclear copy number (relative to mtDNA copies)

are among the potential explanations for this failure. We

are aware of only two population genetic analyses of

macrobial eDNA, and both targeted humans. Kapoor et al.

(2014) applied Ion Torrent sequencing to human mtDNA

control region amplicons from urban Cincinnati, USA

streams, and Afshinnekoo et al. (2015a) applied Illumina

shotgun sequencing to eDNA from object surfaces in the

New York City, USA subway system. These studies have

demonstrated that macrobial eDNA can provide data from

rapidly evolving loci, in both organellar and nuclear

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123

genomes, which coarsely describe geographic patterns of

population genetic diversity. Applying similar approaches

to wildlife conservation may require considerable popula-

tion genetic reference data but remains a realistic and

exciting possibility.

Functional genetics and genomics

The decreasing cost of biotechnology, in particular HTS,

has enabled functional genomic analysis of species of

conservation interest using approaches previously limited to

model organisms (Steiner et al. 2013). Practical applica-

tions include identification of adaptive or fitness-related

loci, monitoring loci related to stress events, and describing

the molecular basis of inbreeding depression (Schwartz

et al. 2007; Paige 2010). Mitochondrial DNA from one

species can comprise just 10-8 percent of the total eDNA in

freshwater (Turner et al. 2014a); thus, low template quantity

and background interference present challenges for func-

tional genomic analysis of macrobial eDNA, particularly

for single-copy nuclear loci (Morin et al. 2007). However,

paleogenetic studies have demonstrated that these types of

functional genomic data can be obtained from biomaterial

exposed to substantial DNA/RNA decay (Fordyce et al.

2013). Furthermore, microbial metagenomics has paved the

way for functional genomic analysis of eDNA, demon-

strating how to analyze functional genomic data from

complex environmental mixtures of individuals and species

(Mendoza et al. 2015). In principle, nothing prevents sim-

ilar applications for macrobial eDNA, but we are not aware

of any studies which have demonstrated functional genomic

analysis for macrobiota from degraded, extraorganismal

DNA or RNA in environmental mixtures.

Progress in eDNA-based functional genomics will likely

follow two complementary paths: (1) using a priori

knowledge of functional genomic loci to target specific

DNA or RNA fragments in environmental samples (Jae-

nicke-Despres et al. 2003) and (2) using shotgun

sequencing of total eDNA or environmental RNA (eRNA)

with bioinformatic assignment to taxa, genomes, and

functions (Su et al. 2014). The first path is immediately

accessible to studies using genomic model organisms and

sampling designs with predictable and robust differences in

genome function (e.g., Robinson et al. 2012). This path will

expand to conservation as genomic resources develop for

threatened and invasive species (Shafer et al. 2015). The

second path is challenged by the large, complex,

endosymbiotic, and gene-sparse genomes of eukaryotes

compared to prokaryotes (reviewed in Gilbert and Dupont

2011). Nevertheless, accumulation of assembled macrobial

genomes (Koepfli et al. 2015) will ease this challenge, and

macrobe-inclusive metagenomic analyses of eDNA and

eRNA are advancing the necessary bioinformatic tools

(Mendoza et al. 2015). Molecules containing functional

genomic information from macrobiota are clearly present

in environmental samples. For example, Orsi et al. (2013)

reported extraorganismal arthropod RNA from anoxic sub-

seafloor sediments over 104 years old. Using functional

eDNA/eRNA data for wildlife conservation will require

substantial investment to overcome the technical hurdles,

but converging advances from many fields inspire opti-

mism along this frontier.

Alternative environmental samples

As eDNA applications grow to include the frontiers we

have described above and additional objectives, so too will

interest in applying these technologies in novel conditions

and environments. These developments will necessitate

creative new approaches to eDNA collection. We have

already begun to see imaginative approaches to what

constitutes an environmental sample. For example, snow

samples containing urine have been used for species and

individual identification of French wild canids (Valiere and

Taberlet 2000), and traces of saliva left on browsed twigs

have been used to identify ungulate species (Nichols et al.

2012). eDNA methods have been applied to bulk-collected

carrion flies, not in a study of the flies themselves, but

rather as a tool to assess local mammalian biodiversity

(Calvignac-Spencer et al. 2013). Similarly, spider web can

serve as an eDNA source for both the spider and its prey

(Xu et al. 2014). Human eDNA can be found in settled dust

and the bodies of small household insects (Toothman et al.

2008; Kester et al. 2010). Researchers have also assessed

benthic invertebrate biodiversity by sampling preservative

ethanol (Hajibabaei et al. 2012). Although preservative

ethanol is not an environmental sample per se, we note the

work of Hajibabaei et al. (2012) and others using non-

destructive DNA sampling methods (e.g. Thomsen et al.

2009) with excitement in the present section because of the

potential for this eDNA-inspired method to enable genetic

analysis of museum specimens and other archived samples

from the past which may benefit contemporary conserva-

tion challenges and ecological understanding.

Remote and autonomous sampling and analysis

Improving technology to enable remote and autonomous

(i.e. without need of constant human input/supervision)

eDNA methodologies will greatly expand the potential of

eDNA applications to benefit conservation. Already, vast

oceanic distances and depths have led marine microbiolo-

gists to develop robotic systems for collection of eDNA

samples (Scholin 2010). The most sophisticated platform

autonomously conducts water filtration, DNA extraction,

and genetic assays while deployed and transmits data via

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satellite (Preston et al. 2011). Surveillance of airborne

biological hazards has motivated similar developments

(Hindson et al. 2005), and engineers have also demon-

strated use of unmanned aerial systems (i.e. ‘drones’) to

remotely collect water samples (Ore et al. 2015). In prin-

ciple, all of these devices are currently applicable to

macrobiota, and one air sampling study has reported suc-

cessful detection of eDNA from diverse animals and plants

(Yooseph et al. 2013). Several systems are already com-

mercially available (e.g. http://www.mclanelabs.com);

however, high purchase and operating costs may limit

dedicated use for conservation efforts. Post-hoc macrobial

analysis of microbe-motivated samples from these systems

likely represents the first step toward broadening their use

across taxa and disciplines. Furthermore, the generic nature

of some environmental samples, such as water, makes

eDNA sampling amenable to automation, and opportunities

abound for integrating genomic data collection and anal-

ysis with remote sensing technologies that traditionally

focus on electromagnetic, acoustic, or other data. These

same features may also open the door to conservation and

research efforts at unprecedented geographic scales by

engaging citizen scientists to aid with environmental

sample collection (Biggs et al. 2014).

Isolating molecules other than DNA

from environmental samples

The field of ecology may be on the cusp of a transformation

based on the emergence of metagenomics technologies;

Poole et al. (2012) have coined the term ‘‘ecosystomics’’ to

describe the upcoming flood of affordable, easily-collected

genetic information about individuals, populations, and

communities. Our review—and indeed most of the research

relevant to the scope of this review to date—has focused on

detecting DNA within the environment as an indicator of

species presence. However, other biological molecules

may also offer opportunities for conservation and research.

For example, RNA is generally less stable than DNA, and

for this reason its degradation has been used by forensic

scientists to estimate the time since deposition of biological

material (Bremmer et al. 2012). Therefore, assays targeting

eRNA may provide narrower spatiotemporal inferences of

organism presence or abundance than eDNA. Forensic

scientists have used messenger RNA (mRNA) to identify

different body fluids left at crime scenes (Vennemann and

Koppelkamm 2010), which suggests mRNA could provide

information about the physiological origin of biomaterial in

environmental samples. Microbial metatranscriptomics

provides guidance for using HTS technologies to charac-

terize eRNA (Shi et al. 2009). Quantifying proteins in the

environment combined with a knowledge of proteomics

may be useful as an indicator of organismal activity or

ecosystem health, as the activation of certain genes could

indicate responses to environmental stimuli. Microbial

metaproteomics has demonstrated how community-scale

analysis of environmental proteins can be accomplished

(Maron et al. 2007), and the collection and analysis of

intact polar membrane lipids has been used to describe

microbial community composition in sediment and water

column samples (Schubotz et al. 2009). Thus, DNA may

soon represent one of many biological materials collected

and analyzed from environmental samples.

Conclusion

eDNA analysis represents an emerging tool for research

and conservation. Already, eDNA tools have been applied

in diverse systems, including terrestrial, freshwater, and

marine environments. The principles of detecting genetic

material within environmental samples were first utilized in

the field of microbiology, and today similar techniques

continue to be expanded to include an ever-increasing

breadth of macrobial organisms. As technology continues

to rapidly improve and costs decline, the list of potential

future conservation and research applications of eDNA is

genuinely exciting. In the present review, we have outlined

the frontiers of conservation-focused eDNA applications in

which we see the most potential for growth, including the

use of eDNA for census or measuring biomass of popula-

tions, population genetic and genomic analyses via eDNA,

inclusion of other indicator biomolecules such as envi-

ronmental RNA or proteins, and expanded technology

enabling automation of sample collection and analysis as

well as sampling of a wide array of creative environmental

samples. However, a more complete understanding of what

we have termed the ecology of eDNA—its origin, state,

transport, and fate within the environment—is needed to

inform eDNA collection and analysis as well as maximize

the research and conservation potential of future eDNA

applications (Figs. 1, 2). The application of eDNA-based

technology is already beginning to influence conservation,

management, and policy decisions, despite existing

uncertainty (Kelly et al. 2014a). Nevertheless, there is

much work to be done to continue close the gap between

research and management considering the application and

interpretation of genetic methods including eDNA appli-

cations (Darling 2015), and the present review has pro-

vided a framework to guide research and organize

understanding as eDNA frontiers continue to advance.

Acknowledgments Thank you to Sean Hoban for encouragement to

prepare this review. Comments from Sean Hoban and three anony-

mous reviewers greatly improved the manuscript. The ideas presented

herein have benefited from discussions with many individuals, espe-

cially Robert Everhart, Christopher Jerde, and David Lodge. Rebecca

Conserv Genet

123

Turner assisted with figures. Figure icons used under CC BY 3.0

license are from Mallory Hawes (fish), Olivier Guin (test tube), Chris

Dawson (wave), and Pratyush Tewari (hourglass). CRT was sup-

ported by NSF IGERT grant award #0504495 to the GLOBES

graduate training program at the University of Notre Dame. Mention

of specific products or trade names have been for illustrative purposes

only and should not be considered as an endorsement.

Open Access This article is distributed under the terms of the

Creative Commons Attribution 4.0 International License (http://crea

tivecommons.org/licenses/by/4.0/), which permits unrestricted use,

distribution, and reproduction in any medium, provided you give

appropriate credit to the original author(s) and the source, provide a

link to the Creative Commons license, and indicate if changes were

made.

Appendix: eDNA techniques and implicationsfor conservation genetics

Our focus on the ecology of eDNA puts a comprehensive

discussion of techniques beyond the scope of this paper.

However, thoughtful selection and transparent presentation

of eDNA methods represent important considerations as

eDNA applications and the field of researchers exploring

the ecology of eDNA continue to expand. Here we briefly

describe a few examples.

Study design

Eukaryotes have up to three separate genomes: nuclear,

mitochondrial, and plastid. The structure, function, inheri-

tance, and evolution of these genomes varies across the tree

of life (e.g. Zouros et al. 1994) and creates important dif-

ferences for their use in identifying species or populations

from DNA sequence (Steele and Pires 2011). In addition to

these features, eDNA studies need to consider the

anatomical location and physiological shedding of different

genomes in target taxa. For example, the quantity of

chloroplasts in angiosperm pollen varies across species,

making the choice of plastid or nuclear genomes important

for studies of plant eDNA in air, sediment, or pollinator

forage (Bennett and Parducci 2006; Kraaijeveld et al. 2015;

Richardson et al. 2015). Metazoan eDNA studies have

tended to use mitogenome regions because of the higher

number of mitochondrial versus nuclear genomes per cell

(Rees et al. 2015); however, eukaryotic ribosomal DNA

copy number per nuclear genome can reach 19,300 in

animals and 26,048 in plants (Prokopowich et al. 2003)—

numbers which are comparable to the largest numbers of

mitogenomes per cell (e.g., 22,000; Caldwell et al. 2011).

The value of mtDNA for eDNA studies may thus have

more to do with its structural resistance to degradation than

its cellular copy number (discussed in Turner et al. 2014a).

Using multiple regions from one or more genomes can

improve target detection (Evans et al. 2015).

Sample collection and preparation

Macrobial DNA can be found almost everywhere, but the

behavior of target organisms, ecology of eDNA, and study

goals determine which methods are optimal for collecting

and preparing samples. For example, stream mtDNA con-

centration from a fully aquatic amphibian was significantly

higher in breeding season than other months (Spear et al.

2015). Increased eDNA production from gamete release

and lower DNA degradation in intact gametes are likely

explanations. Thus, an eDNA survey to identify repro-

ducing populations might include high-frequency temporal

sampling and water filter pore size matched to gamete size.

Optimal preservation and extraction of target DNA from

environmental samples will also vary across taxa and

environments. For example, pollen DNA extraction may

require crushing pollen grains (Kraaijeveld et al. 2015),

and total sediment DNA extraction benefits from chemical

desorption of sediment-adsorbed DNA molecules (Yank-

son and Steck 2009). Thus, a ‘‘one size fits all’’ approach is

not feasible for collecting and preparing eDNA samples,

and attention to organismal and environmental idiosyn-

crasies should inform methodological choices.

eDNA assays

Narrow assays target one species or genus using DNA

amplification technology such as real-time quantitative

PCR or digital PCR (Doi et al. 2015). Oligonucleotides are

designed to exclusively amplify the taxon, allowing con-

clusions about target DNA presence and quantity imme-

diately after amplification (Turner et al. 2014b). Broad

assays use high-throughput sequencing (HTS) or microar-

rays to target large clades such as flowering plants or cel-

lular organisms (Vuong et al. 2013). DNA amplification or

hybridization can enrich for a target clade prior to

sequencing, or direct shotgun sequencing can be used to

generate sequence data from all organisms (Shokralla et al.

2012). Post-sequencing bioinformatic analysis provides

conclusions about target DNA presence and quantity. A

holistic consideration of the ecology of eDNA (Fig. 1)

shows how various processes can produce detection or non-

detection, thus rigorous precautions and quality controls

are needed to reduce uncertainty(Murray et al. 2015).

Empirical studies of forensic DNA mixture interpretation

demonstrate how observer effects (i.e. the human tendency

to interpret data in a manner consistent with one’s expec-

tations) create subjectivity and bias (Dror and Hampikian

Conserv Genet

123

2011), and users of eDNA assays may benefit from con-

sidering this phenomenon.

Analysis of eDNA data

Bioinformatic analysis of HTS data from eDNA samples

represents a complex and rapidly changing landscape

(Edgar and Flyvbjerg 2015). Macrobial eDNA studies use

massive datasets to evaluate low abundance sequences

from unseen taxa, thus susceptibility to artifacts such as

sample contamination (Lusk 2014), amplification errors

(Schnell et al. 2015), sequencing errors (Robasky et al.

2014), computational artifacts (Rossberg et al. 2014), and

inaccurate taxonomic assignment (Afshinnekoo et al.

2015b), is high. Mendoza et al. (2015) provide a useful

overview for navigating these challenges. From our own

experience applying HTS to assay macrobial eDNA, we

recommend using negative controls with carrier biomate-

rial (Xu et al. 2009) and positive controls with mock

community DNA (Schloss et al. 2011). Different perspec-

tives are still emerging about how control data should be

used to ‘‘correct’’ co-sequenced eDNA data (Nguyen et al.

2015), but the transparent and self-critical cognitive

approach recommended by Gilbert et al. (2005) for ancient

DNA is highly applicable to all eDNA studies, particularly

those with conservation applications where substantial

economic and legal consequences may result (Kelly 2014).

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